According to the free radical theory of aging, the cumulative molecular damages inflicted by the byproducts of energy metabolism produced by mitochondria during oxidative phosphorylation lead to a loss of cellular function. The capacity of a cell to generate ATP is dependent upon both its rate of glycolysis and mitochondrial respiration. In general, it is the ability of the tissue to re- oxidize NADH, whether by respiratory mechanisms (reduction of oxygen to water) or by glycolytic mechanisms (reduction of pyruvate to lactate), that determines the capacity of cells to generate ATP. It has been shown that human cells that completely lack their mitochondrial DNA can grow without mitochondrial respiratory function, provided excess pyruvate and uridine are present in the growth medium. It has also been demonstrated that cells in culture can grow anaerobically on a glucose medium provided excess pyruvate is added to the growth medium. The glycolytic pathway can thereby function continuously to provide sufficient ATP for cell growth. Using normal human diploid fibroblasts and cells depleted of their mtDNA (i.e., lacking a functional respiratory chain), the proposed research project aims at studying the dual contribution of the two cellular energy-generating pathways and defining the point at which energy production is optimized. It will be done by investigating separately as well as concomitantly the role played by the two parameters, oxygen and pyruvate, in influencing the cellular replicative lifespan. The specific aims of the project are: 1) to generate three different mtDNA-less cell line from three different human diploid fibroblast cell lines; 2) to measure the replicative capability of the normal and rho0 derivatives cell lines grown under low oxygen tension at various time of their lifespan in order to correlate oxygen pressure and cellular replicative potential; and 3) to evaluate the role of the energetic substrate, pyruvate, on the proliferative lifespan of the three human normal diploid fibroblast lines and their derivative rho0 lines in order to test whether the combined effect of high pyruvate and low oxygen can enhance the energetic level of the cells resulting in a longer replicative lifespan.